The proposed research will concentrate on the mechanism of action of hormones on hepatic gluconeogenesis. The hypothesis to be tested is as follows: Hormonal control of hepatic gluconeogenesis is mediated by cAMP dependent and/or independent phosphorylation of cytoplasmic enzymes involved in the F-6-P/FDP and PEP/Pyruvate substrate cycles. The research will focus on L-pyruvate kinase and FDPase as sites of hormone action since flux through and activity of these enzymes has been shown to be under hormonal control. The effect of glucagon, insulin and catecholamines on flux through these enzymes in intact cells will be characterized. These studies will be correlated with studies of the effect of these various hormones on 32P incorporation into pyruvate kinase and FDPase. The labeled enzymes will be isolated by immunological methods. The phosphorylation state of the enzyme in intact cells will be correlated with the enzyme activity measured in hepatocyte homogenates and with cAMP levels. The effect of CA 2 ion on these parameters will also be evaluated. Furthermore, the kinases and phosphatases which are presumably responsible for modification of these enzymes in liver will be isolated and characterized, particularly with regard to cAMP dependency, and their control by hormones studied. Isolated hepatocytes will be used as the experimental tool in these studies. It should be possible to determine if the effects of hormones on the gluconeogenic pathway are mediated by phosphorylation-dephosphorylation of specific liver proteins and whether or not these changes correlate with changes in cAMP and CAMP-dependent protein kinase activation.